Marathon running transiently increases c-Jun NH2-terminal kinase and p38 activities in human skeletal muscle

Abstract

We examined the pattern of activation and deactivation of the stress-activated protein kinase signalling molecules c-Jun NH2-terminal kinase (JNK) and p38 kinase in skeletal muscle in response to prolonged strenuous running exercise in human subjects. Male subjects (n = 14; age 32 +/- 2 years; VO2,max 60 +/- 2 ml kg-1 min-1) completed a 42.2 km marathon (mean race time 3 h 35 min). Muscle biopsies were obtained 10 days prior to the marathon, immediately following the race, and 1, 3 and 5 days after the race. The activation of JNK and p38, including both p38alpha and p38gamma, was measured with immune complex assays. The phosphorylation state of p38 (alpha and gamma) and the upstream regulators of JNK and p38, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 6 (MKK6), were assessed using phosphospecific antibodies. JNK activity increased 7-fold over basal level immediately post-exercise, but decreased back to basal levels 1, 3 and 5 days after the exercise. p38gamma phosphorylation (4-fold) and activity (1.5-fold) increased immediately post-exercise and returned to basal levels at 1, 3 and 5 days following exercise. In contrast, p38alpha phosphorylation and activity did not change over the time course studied. MKK4 and MKK6 phosphorylation increased and decreased in a trend similar to that observed with JNK activity and p38gamma phosphorylation. Prolonged running exercise did not affect JNK, p38alpha, or p38gamma protein expression in the days following the race. This study demonstrates that both JNK and p38 intracellular signalling cascades are robustly, yet transiently increased following prolonged running exercise. The differential activation of the p38 isoforms with exercise in human skeletal muscle indicates that these proteins may have distinct functions in vivo.

Figure 1. Effect of marathon running on JNK activity

A, reaction products from the JNK activity assay from one representative subject in the basal state (B), immediately post-exercise (Post), and days 1, 3 and 5 post-exercise (D1, D3, D5). B, JNK activity for all subjects completing the marathon race. Data are means ±s.e.m., *P < 0.05vs. basal state and days 1, 3 and 5. C, representative immunoblot of JNK protein expression in muscle lysates from one representative subject.

Figure 2. Differential regulation of p38α and p38γ phosphorylation immediately following a marathon race

A, immunoblot of p38α and p38γ phosphorylation from one representative subject in the basal state (B), immediately post-exercise (Post), and days 1, 3 and 5 (D1, D3, D5) post-exercise. B, p38γ phosphorylation for all subjects completing the marathon race. Data are means ±s.e.m., *P < 0.05vs. basal value and days 1, 3 and 5. C, representative immunoblot of p38γ protein expression in muscle lysates from one representative subject.

Figure 3. Effect of marathon running on p38α and p38γ activity

A, reaction products from the p38α and p38γ activity assays from four representative subjects in the basal state (B) and immediately post-exercise (Post). B, p38γ activity for all but one subject completing the marathon race (n = 13). Data are means ±s.e.m., *P < 0.05vs. basal values.

Figure 4. Effect of marathon running on MKK4 and MKK6 phosphorylation

Representative immunoblots of MKK4 and MKK6 phosphorylation are shown at the top of each figure. A, MKK4 phosphorylation under basal conditions (B), immediately post-exercise (Post), and days 1, 3 and 5 (D1, D3, D5) post-exercise. B, MKK6 phosphorylation under basal conditions (B), immediately post-exercise (Post), and days 1, 3 and 5 (D1, D3, D5) post-exercise. Data are means ±s.e.m., *P < 0.05vs. basal levels and days 1, 3 and 5.

Figure 5. Correlation between JNK activity and p38γ phosphorylation

Correlation analysis was determined on the fold-increases in JNK activity and p38γ phosphorylation immediately post-exercise for all subjects (n = 14).